Human CD1d consists of 5 genes (CD1A-E) that are encoded on chromosome 1 outside the MHC locus on chromosome 6. Human CD1d has strong structural similarities to MHC class I but also shows several features in common with MHC class II, suggesting a distinct relationship to both of the previously characterized classical MHC molecules. CD1d, a member of this family, is prominently expressed in hepatocytes and on the cell surface of intestinal epithelial cells (IEC). Although the exact role of CD1d in the mucosal immune system is unclear, CD1d exhibits unique biochemical properties and functional characteristics. We have previously shown that CD1d is expressed on the cell surface of normal IECs as a 37-kD, nonglycosylated molecule independently of beta2-microglobulin which is clearly distinct from all other MHC class I-related molecules. In addition, we have shown that this biochemical form may be directly involved in the proliferation of T cells to normal IECs. In order to understand the biosynthesis, assembly and function of this distinct antigen-presenting molecule, it will be important to define the proteins that interact with CD1d. The goal of this proposal is, therefore, to identify and characterize CD1d interacting intracellular proteins by 1) screening a cDNA library from human hepatocytes using the yeast two-hybrid system and biosynthetic labeling of IEC cell lines and a CD1d transfected model system using CD1d specific antibodies, and identify CD1d interacting extracellular proteins by 2) screening a cDNA library of T cells using the yeast two-hybrid system and examining interactions of T cell proteins with an Fc-CD1d fusion protein.